All eyes show an association of photoreceptors with dark pigment. In vertebrates, this association can be traced back to the action of the bHLH-Zip transcription factor MITF, which controls the generation of melanin-bearing pigment cells, and the paired-domain transcription factor PAX6, which controls the generation of photoreceptors in the retina. High level expression of these transcription factors is usually mutually exclusive, except in the neuroepithelium-derived retinal pigment epithelium (RPE) where they are co-expressed during development. We approached the question of the role of PAX6 in the RPE genetically, using a variety of mouse Mitf and Pax6 alleles individually and in combinations. The results show that a reduction in the level of functional PAX6 protein exacerbates the dorsal RPE-to-retina transition normally associated with Mitf mutations, and that overexpression of PAX6 protein from a yeast artificial chromosome transgene alleviates this Mitf-mediated RPE-to-retina transition. Hence, the results suggest that in the RPE, Pax6 has an anti-retinogenic role, in contrast to its pro-retinogenic role in the retina. To address the question of the molecular pathways involved in this anti-retinogenic activity of Pax6, we use a candidate gene approach, employing RT-PCR to obtain temporal and spatial gene expression data at the mRNA level and immunocytochemistry to obtain corresponding data at the protein level. Moreover, we use chromatin immunoprecipitation procedures on dissected embryonic eye tissues to establish transcription factor interactions with the regulatory regions of potential target genes. These studies should help us to determine the molecular mechanisms of RPE versus retina development and give insights into the evolutionarily conserved use of Mitf and Pax6 and its homologs in eye development from jellyfish to man.